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1.
The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2''s loading to the site of procentriole formation. Four conserved serines in Ana2''s STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously. 相似文献
2.
Burcu Inan? Monika Pütz Pierce Lalor Peter Dockery Ryoko Kuriyama Fanni Gergely Ciaran G. Morrison 《Molecular biology of the cell》2013,24(17):2645-2654
Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase–arrested cells. 相似文献
3.
Naomi R. Stevens Jeroen Dobbelaere Kathrin Brunk Anna Franz Jordan W. Raff 《The Journal of cell biology》2010,188(3):313-323
In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP—orthologues of ZYG-1, SAS-6, and SAS-4, respectively—are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery. 相似文献
4.
Hatch EM Kulukian A Holland AJ Cleveland DW Stearns T 《The Journal of cell biology》2010,191(4):721-729
Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1-217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation. 相似文献
5.
Brian J. Galletta Katherine C. Jacobs Carey J. Fagerstrom Nasser M. Rusan 《The Journal of cell biology》2016,213(4):435-450
Centrioles are the foundation of two organelles, centrosomes and cilia. Centriole numbers and functions are tightly controlled, and mutations in centriole proteins are linked to a variety of diseases, including microcephaly. Loss of the centriole protein Asterless (Asl), the Drosophila melanogaster orthologue of Cep152, prevents centriole duplication, which has limited the study of its nonduplication functions. Here, we identify populations of cells with Asl-free centrioles in developing Drosophila tissues, allowing us to assess its duplication-independent function. We show a role for Asl in controlling centriole length in germline and somatic tissue, functioning via the centriole protein Cep97. We also find that Asl is not essential for pericentriolar material recruitment or centrosome function in organizing mitotic spindles. Lastly, we show that Asl is required for proper basal body function and spermatid axoneme formation. Insights into the role of Asl/Cep152 beyond centriole duplication could help shed light on how Cep152 mutations lead to the development of microcephaly. 相似文献
6.
Joseph E. Klebba Brian J. Galletta Jonathan Nye Karen M. Plevock Daniel W. Buster Natalie A. Hollingsworth Kevin C. Slep Nasser M. Rusan Gregory C. Rogers 《The Journal of cell biology》2015,208(4):401-414
Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification. 相似文献
7.
Plk4-induced centriole biogenesis in human cells 总被引:9,自引:0,他引:9
Kleylein-Sohn J Westendorf J Le Clech M Habedanck R Stierhof YD Nigg EA 《Developmental cell》2007,13(2):190-202
We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells. 相似文献
8.
Lauren K. Slevin Erin M. Romes Mary G. Dandulakis Kevin C. Slep 《The Journal of biological chemistry》2014,289(30):20727-20739
Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156–251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ∼34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis. 相似文献
9.
Thomas L. Steinacker Siu-Shing Wong Zsofia A. Novak Saroj Saurya Lisa Gartenmann Eline J.H. van Houtum Judith R. Sayers B. Christoffer Lagerholm Jordan W. Raff 《The Journal of cell biology》2022,221(9)
Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process—indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis. 相似文献
10.
Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly
Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process. 相似文献
11.
A Proximal Centriole-Like Structure Is Present in Drosophila Spermatids and Can Serve as a Model to Study Centriole Duplication
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Stephanie Blachon Xuyu Cai Kela A. Roberts Kevin Yang Andrey Polyanovsky Allen Church Tomer Avidor-Reiss 《Genetics》2009,182(1):133-144
Most animals have two centrioles in spermatids (the distal and proximal centrioles), but insect spermatids seem to contain only one centriole (Fuller 1993), which functionally resembles the distal centriole. Using fluorescent centriolar markers, we identified a structure near the fly distal centriole that is reminiscent of a proximal centriole (i.e., proximal centriole-like, or PCL). We show that the PCL exhibits several features of daughter centrioles. First, a single PCL forms near the proximal segment of the older centriole. Second, the centriolar proteins SAS-6, Ana1, and Bld10p/Cep135 are in the PCL. Third, PCL formation depends on SAK/PLK4 and SAS-6. Using a genetic screen for PCL defect, we identified a mutation in the gene encoding the conserved centriolar protein POC1, which is part of the daughter centriole initiation site (Kilburn et al. 2007) in Tetrahymena. We conclude that the PCL resembles an early intermediate structure of a forming centriole, which may explain why no typical centriolar structure is observed under electron microscopy. We propose that, during the evolution of insects, the proximal centriole was simplified by eliminating the later steps in centriole assembly. The PCL may provide a unique model to study early steps of centriole formation.THE centriole is a cylindrical structure rich in microtubules, which are organized in a ninefold symmetry. As the template of the ciliary axoneme, the centriole transmits its symmetry to the cilium. Dividing cells contain two centrosomes at the cell poles, each containing a pair of centrioles (mother and daughter centrioles) surrounded by a thick layer of pericentriolar material (PCM). Upon differentiation, the mother centriole of each pair becomes a basal body, which acts as a template for the cilium (Azimzadeh and Bornens 2007). The function of the daughter centriole is less clear. For example, in animal spermatids, the mother centriole, known as the distal centriole, becomes a basal body and gives rise to the sperm flagellum (Krioutchkova and Onishchenko 1999; Sathananthan et al. 2001). The daughter centriole in spermatids, known as the proximal centriole, is attached to the nucleus.Unlike other animal groups, multiple ultrastructural studies of insect sperm find only one centriole that has the canonical structure of microtubules organized in a ninefold symmetry (Anderson 1967; Tates 1971; Tokuyasu 1975a,b). This centriole forms the flagellum and is therefore the homolog to the vertebrate distal centriole. During spermatid differentiation (Figure 2A), a structure, called the centriolar adjunct (CA), appears transiently around this centriole (Friedlander and Wahrman 1971; Tates 1971; Tokuyasu 1975a,b; Wilson et al. 1997). The centriolar adjunct is a very dynamic structure, which shrinks during spermatid differentiation to form a collar around the distal centriole and then disappears (Tates 1971; Wilson et al. 1997). Studies using light microscopy found that γ-tubulin localizes and redistributes around the centriole in a way similar to the CA (Wilson et al. 1997), suggesting that it may serve as its marker. Earlier electron microscopy (EM) studies describe the appearance of a centriole inside the centriolar adjunct after it obtains the collar-shape structure (Anderson 1967; Phillips 1970). But this structure is amorphous and does not exhibit the morphological features of a centriole. Interestingly, the earliest intermediate observed during centriole formation is described as an amorphous structure (Anderson 1967; Dippell 1968; Sorokin 1968; Allen 1969).Open in a separate windowFigure 2.—Ana1 labels a novel structure appearing near the mother centriole in spermatids. (A) Diagram depicting the different stages of spermatid development based on the observations of Tates (1971). (M, mitochondria; N, nucleus; Ax, axoneme). The basal body or giant centriole (Cen) is surrounded by the centriolar adjunct (CA) and, near it, we can follow the formation of the PCL. (B) We use phase-contrast pictures (unfixed testis) to determine the spermatid stage. The onion stage (stage S13) is characterized by a round nucleus (N) of the same size as the mitochondrial derivatives (M). The cell body of intermediate spermatids (stages 15 and 16) elongates, forming short protrusions (arrows), but the nucleus remains round. In late spermatid development (stage 17), the nucleus becomes oval. Ana1-GFP labels the giant centriole (Cen), and in intermediate spermatids a bulge forms on one side and becomes individualized as PCL in late spermatid development. (C) Staining with anti-γ-tubulin antibody shows that the PCL labeled by Ana1 is an entity different from the γ-tubulin collar that is reminiscent of the centriolar adjunct (CA). (D) Antibody against Ana1 labels the V-shape pair of giant centrioles in primary spermatocytes (left) and the giant centriole and PCL in spermatids (right) in flies expressing Ana1-GFP. (E) In wild-type primary spermatocytes, anti-Ana1 antibody stains the endogenous protein in the giant centrioles and colocalizes with γ-tubulin staining (left). In spermatids, the antibody labels the PCL, demonstrating that its formation is not due to centriolar protein overexpression (right).Centriole duplication provides the cell with a mechanism for tightly controlling the number of centrosomes and cilia. In most cells, the centriole duplicates once per cell cycle and a single new centriole is formed in the vicinity of each mother centriole. The mechanism ensuring that only one daughter centriole forms in the vicinity of the mother centriole is not known (Strnad and Gonczy 2008). Two major limiting factors hinder the investigation of this process: (1) the difficulty of distinguishing between the mother centriole and the forming daughter centriole and (2) the short time that it takes for the process to reach completion, which in turn hinders the identification of intermediates. Few model systems are currently available for studying this process (Pelletier et al. 2006; Kleylein-Sohn et al. 2007).Here, we demonstrate that fly spermatids contain a novel structure that is labeled by centriolar proteins and that forms in the vicinity of the proximal end of the mother centriole. Because it is reminiscent of the vertebrate proximal centriole but no morphological signatures of a centriole have been observed, we propose to call it proximal centriole-like (PCL). While studying the pan-centriolar protein Ana1, we found that it labeled the PCL. The PCL forms before γ-tubulin is redistributed as a collar, showing that it is a distinct entity. We then found that the formation of the PCL depends on the proteins SAK/PLK4 and SAS-6, which are essential early in daughter centriole formation, but not on SAS-4, which in worms is required later in the process. These observations indicate that the PCL represents an early intermediate structure in centriole formation. We also tested the involvement of the centriolar protein Bld10p/Cep135, which was found in Chlamydomonas and humans to be a component of the centriole cartwheel and wall (Hiraki et al. 2007; Kleylein-Sohn et al. 2007). We found that Bld10p is recruited to the PCL only later in the process and is not required for PCL formation. We performed a genetic screen finding that the Drosophila ortholog of POC1 is essential for the formation of normal PCL. POC1 was identified previously in a proteomic screen as a centriolar protein and is localized to the early intermediate structure in centriole/basal body formation (Keller et al. 2005, 2008; Kilburn et al. 2007). We propose to use PCL formation as a model to study the molecular pathway for centriole initiation. Our results suggest that POC1, like PLK4 and SAS-6, plays an important role early in centriole formation whereas Bld10p function is required later as SAS-4 is. 相似文献
12.
Cizmecioglu O Arnold M Bahtz R Settele F Ehret L Haselmann-Weiss U Antony C Hoffmann I 《The Journal of cell biology》2010,191(4):731-739
Both gain and loss of function studies have identified the Polo-like kinase Plk4/Sak as a crucial regulator of centriole biogenesis, but the mechanisms governing centrosome duplication are incompletely understood. In this study, we show that the pericentriolar material protein, Cep152, interacts with the distinctive cryptic Polo-box of Plk4 via its N-terminal domain and is required for Plk4-induced centriole overduplication. Reduction of endogenous Cep152 levels results in a failure in centriole duplication, loss of centrioles, and formation of monopolar mitotic spindles. Interfering with Cep152 function prevents recruitment of Plk4 to the centrosome and promotes loss of CPAP, a protein required for the control of centriole length in Plk4-regulated centriole biogenesis. Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process. 相似文献
13.
Kei K. Ito Koki Watanabe Haruki Ishida Kyohei Matsuhashi Takumi Chinen Shoji Hata Daiju Kitagawa 《The Journal of cell biology》2021,220(3)
Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation. 相似文献
14.
Gang Dong 《Open biology》2015,5(8)
Centrioles are short microtubule-based organelles with a conserved ninefold symmetry. They are essential for both centrosome formation and cilium biogenesis in most eukaryotes. A core set of five centriolar proteins has been identified and their sequential recruitment to procentrioles has been established. However, structures at atomic resolution for most of the centriolar components were scarce, and the underlying molecular mechanisms for centriole assembly had been a mystery—until recently. In this review, I briefly summarize recent advancements in high-resolution structural characterization of the core centriolar components and discuss perspectives in the field. 相似文献
15.
Granule neuron progenitors (GNPs) are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh) secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3), another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis. 相似文献
16.
Chad G. Pearson Daniel P.S. Osborn Thomas H. Giddings Jr. Philip L. Beales Mark Winey 《The Journal of cell biology》2009,187(6):905-920
Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T.
thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis. 相似文献
17.
Yusuke Mori Yoko Inoue Sayori Tanaka Satoka Doda Shota Yamanaka Hiroki Fukuchi Yasuhiko Terada 《PloS one》2015,10(10)
The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC) onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs). Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM) around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding–deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2. 相似文献
18.
Kerstin N. Schmidt Stefanie Kuhns Annett Neuner Birgit Hub Hanswalter Zentgraf Gislene Pereira 《The Journal of cell biology》2012,199(7):1083-1101
Cilia formation is a multi-step process that starts with the docking of a vesicle at the distal part of the mother centriole. This step marks the conversion of the mother centriole into the basal body, from which axonemal microtubules extend to form the ciliary compartment. How vesicles are stably attached to the mother centriole to initiate ciliary membrane biogenesis is unknown. Here, we investigate the molecular role of the mother centriolar component Cep164 in ciliogenesis. We show that Cep164 was indispensable for the docking of vesicles at the mother centriole. Using biochemical and functional assays, we identified the components of the vesicular transport machinery, the GEF Rabin8 and the GTPase Rab8, as interacting partners of Cep164. We propose that Cep164 is targeted to the apical domain of the mother centriole to provide the molecular link between the mother centriole and the membrane biogenesis machinery that initiates cilia formation. 相似文献
19.
David M. Phillips 《The Journal of cell biology》1967,33(1):73-92
Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop giant centrioles with 60–90 singlet tubules disposed in an oval array. Some 9-membered centrioles still may be seen in second instar spermatogonia. Each of these centrioles is associated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles of second instar spermatogonia consist of 20–50 singlet tubules arranged in an oval, sometimes associated with an even larger secondary centriole. The more recently formed centriole of a pair is distinguishable from its partner by a concentric band of electron-opaque material inside its tubules. If a pair of centrioles at right angles to each other is pictured as a "T" formed by two cylinders, the secondary centriole is always the stem of the T; the primary centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a new daughter centriole. A Golgi area of characteristic morphology is found in association with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole may give rise to a daughter morphologically different from itself. Whether the daughter is a 9-membered or giant centriole depends on the tissue type and stage of development. 相似文献
20.
Drosophila asterless and vertebrate Cep152 Are orthologs essential for centriole duplication
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Blachon S Gopalakrishnan J Omori Y Polyanovsky A Church A Nicastro D Malicki J Avidor-Reiss T 《Genetics》2008,180(4):2081-2094
The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl was previously reported to be a centrosomal protein essential for centrosome function. Here we identify mecD, a severe loss-of-function allele of the asl gene, and demonstrate that it is required for centriole and cilia formation. Similarly, Cep152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, we found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication. 相似文献